We have devised a new excitation scheme, which we term red shifted off-resonance large amplitude excitation that can deposit higher amounts of internal energy in ions than conventional resonance excitation. This excitation scheme is effective in fragmenting peptide ions produced by matrix-assisted laser desorption with m/z values up to 3,500. Prior to excitation, it is important to isolate ions of interest with high efficiency. We show here thatisolation efficiencies as high as 100% can be achieved with careful design of the RF scan functions used during ion isolation. In particular, we find that it is important to avoid sudden transitions in the amplitude of the RF field. The combined improvements in the efficiency for ion isolation and the efficacy of ion activation made matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry a practical tool for the characterization of proteins with high sensitivity.